Abstract

Vascular basement membrane is an important structural component of blood
vessels and has been shown to interact with and modulate vascular
endothelial behavior during angiogenesis. During the inductive phase of
tumor angiogenesis, this membrane undergoes many degradative and
structural changes and reorganizes to a native state around newly
formed capillaries in the resolution phase. Such matrix changes are
potentially associated with molecular modifications that include
expression of matrix gene products coupled with conformational changes,
which expose cryptic protein modules for interaction with the vascular
endothelium. We speculate that these interactions provide important
endogenous angiogenic and anti-angiogenic cues. In this report, we
identify an important anti-angiogenic vascular basement
membrane-associated protein, the 26-kDa NC1 domain of theα
1 chain of type IV collagen, termed arresten. Arresten
was isolated from human placenta and produced as a recombinant molecule
in Escherichia coli and 293 embryonic kidney cells. We
demonstrate that arresten functions as an anti-angiogenic molecule by
inhibiting endothelial cell proliferation, migration, tube formation,
and Matrigel neovascularization. Arresten inhibits the growth of two
human xenograft tumors in nude mice and the development of tumor
metastases. Additionally, we show that the anti-angiogenic activity of
arresten is potentially mediated via mechanisms involving cell surface
proteoglycans and the α1β1 integrin on
endothelial cells. Collectively, our results suggest that arresten is a
potent inhibitor of angiogenesis with a potential for therapeutic use.

INTRODUCTION

The development of new blood vessels from preexisting ones is
generally referred to as angiogenesis
(1)
. In the adult,
new blood vessels arise via angiogenesis, a process critical for normal
physiological events such as wound repair, the menstrual cycle, and
endometrium remodeling
(2)
. In the last three decades,
considerable research has been conducted documenting that tumor growth
and metastasis require angiogenesis
(3)
. This process is
pivotal to the survival and subsequent growth of solid tumors beyond a
few cubic millimeters in size
(4)
. Vascular basement
membrane constitutes an insoluble structural wall of newly formed
capillaries and undergoes several changes during tumor-induced
angiogenesis
(5)
. Initially, the membrane is degraded and
disassembled but is finally reorganized to a native state around a
newly formed capillary
(5)
. Such vascular matrix changes
during angiogenesis are associated with the expression of matrix
proteins that can interact with vascular endothelium and provide
endogenous angiogenic and anti-angiogenic signals
(5)
.
Basement membranes are composed of macromolecules such as type IV
collagen, laminin,
HSPGs,
3
fibronectin, and entactin
(6)
. Type IV collagen is
composed of six genetically distinct gene products, namely,α
1–α6(7)
. The α1 andα
2 isoforms are ubiquitously present in human
basement membranes
(8)
. The other four isoforms exhibit
restricted distributions
(9)
. Type IV collagen promotes
cell adhesion, migration, differentiation, and growth
(8)
.
It is thought to play a crucial role in endothelial cell proliferation
and behavior during the angiogenic process
(5)
. Several
studies have shown the anti-angiogenic properties associated with
inhibitors of collagen metabolism, supporting the notion that basement
membrane collagen synthesis and deposition are crucial for blood vessel
formation and survival
(10)
. Additionally, the
COOH-terminal globular NC1 domain of type IV collagen is speculated to
play an important role in the assembly of type IV collagen
suprastructure, basement membrane organization, and modulation of cell
behavior
(11, 12)
. Recently, the NC1 domain of theα
2 chain of type IV collagen (canstatin) was
identified as an angiogenesis inhibitor
(13)
In the
present study, we demonstrate the pivotal role of arresten, the NC1
domain of the α1 chain of type IV collagen, in
modulating the function of capillary endothelial cells and blood vessel
formation using in vitro and in vivo models of
angiogenesis and tumor growth.

MATERIALS AND METHODS

Recombinant Production of Arresten in Escherichia
coli.

The sequence encoding arresten was amplified by PCR from theα
1 NC1 (IV)/pDS vector
(14)
using a forward primer (5′-CGGGATCCTTCTGTTGATCACGGCTTC-3′) and a
reverse primer (5′-CCCAAGCTTTGTTCTTCTCATACAGAC-3′). The resulting cDNA
fragment was digested with BamHI and Hind III and
ligated into predigested pET22b(+) (Novagen, Madison, WI). This placed
arresten downstream of and in frame with the pelB leader sequence,
allowing for periplasmic localization and expression of soluble
protein. Additional vector sequence was added to the protein encoding
amino acids MDIGINSD. The 3′ end of the sequence was ligated in frame
with the polyhistidine tag sequence. Additional vector sequence between
the 3′ end of the cDNA and the his tag encoded the amino acids KLAAALE.
Positive clones were sequenced on both strands.

Plasmid constructs encoding arresten were first transformed into
E. coli HMS174 (Novagen) and then transformed into BL21 for
expression (Novagen). Overnight bacterial culture was used to inoculate
a 500-ml culture in Luria-Bertani medium. This culture was grown for∼
4 h until the cells reached an A600
of 0.6. Then, protein expression was induced by addition of
isopropyl-1-thio-β-d-galactopyranoside
to a final concentration of 1–2 mm. After a 2-h
induction, cells were harvested by centrifugation at 5,000 × g and lysed by resuspension in 6
m guanidine, 0.1 m
NaH2PO4, and 0.01
m Tris-HCl (pH 8.0). Resuspended cells were
sonicated briefly, and centrifuged at 12,000 × g for 30 min. The supernatant fraction was passed over a
5-ml Ni-nitrilotriacetic acid-agarose column (Qiagen,
Chatsworth, CA) four to six times at a speed of 2 ml/min.
Nonspecifically bound protein was removed by washing with both 10 and
25 mm imidazole in 8 m
urea, 0.1 m
NaH2PO4, and 0.01
m Tris-HCl (pH 8.0). Arresten protein was eluted
from the column with increasing concentrations of imidazole (50, 125,
and 250 mm) in 8 m urea,
0.1 m
NaH2PO4, and 0.01
m Tris-HCl (pH 8.0). The eluted protein was
dialyzed twice against PBS at 4°C. A minor portion of the total
protein precipitated during dialysis. Dialyzed protein was collected
and centrifuged at ∼3,500 × g and
separated into pellet and supernatant fractions. Protein concentration
in each fraction was determined by the bicinchoninic acid assay
(Pierce, Rockford, IL) and quantitative SDS-PAGE analysis. The fraction
of total protein in the pellet was ∼22%, with the remaining 78%
recovered as a soluble protein. The total yield of protein was
approximately 10 mg/liter.

Recombinant Production of Endostatin in Yeast.

Mouse endostatin was produced in Picchia pastoris and
purified as described previously
(15)
.

Expression of Arresten in 293 Embryonic Kidney Cells.

We used the pDS plasmid containing α1(IV)NC1
(14)
to PCR amplify arresten in a way that it would add a
leader signal sequence in frame into the pcDNA 3.1 (Invitrogen,
Carlsbad, CA) eukaryotic expression vector. The leader sequence from
the 5′ end of the full-length α1(IV) chain was
cloned 5′ to the NC1 domain to enable protein secretion into the
culture medium. The arresten-containing recombinant vectors were
sequenced using flanking primers. Error-free cDNA clones were further
purified and used for in vitro translation studies to
confirm protein expression (data not shown). The arresten-containing
plasmid and control plasmid were used to transfect 293 cells using the
calcium chloride method. Transfected clones were selected by Geneticin
(Life Technologies, Inc., Gaithersburg, MD) antibiotic treatment. The
cells were passed for 3 weeks in the presence of the antibiotic until
no cell death was evident. Clones were expanded into T-225 flasks and
grown until confluent. Then, the supernatant was collected and
concentrated using an Amicon (Beverly, MA) concentrator. The
concentrated supernatant was analyzed by SDS-PAGE, immunoblotting, and
ELISA for arresten expression. Strong binding in the supernatant was
detected by ELISA (data not shown). The arresten-containing supernatant
was subjected to affinity chromatography using arresten-specific
antibodies
(14)
. Arresten antibody was generated to a
purified protein as described previously
(14)
. This
antibody recognized only the α1 NC1 domain
(14)
. A major peak was identified, containing a monomer of∼
30 kDa that was immunoreactive with arresten antibodies.

Inhibition of Endothelial Cell Proliferation.

CPAE cells were grown to confluence in DMEM with 10% FCS and
kept contact inhibited for 48 h. Human renal cell carcinoma cells
(786-0; data not shown), PC-3 cells (human prostate adenocarcinoma),
HPECs, and A-498 (renal carcinoma) cells (data not shown) were used as
controls in this experiment. Cells were harvested by trypsinization
(Life Technologies) at 37°C for 5 min. A suspension of 12,500 cells
in DMEM with 1% FCS was added to each well of a 24-well plate coated
with 10 μg/ml fibronectin. The cells were incubated for 24 h at
37°C with 5% CO2 and 95% humidity. The medium
was removed and replaced with DMEM containing 0.5% FCS and 3 ng/ml
bFGF (R&D Systems, Inc., Minneapolis, MN). Unstimulated controls
received no bFGF. Cells were treated with concentrations of arresten or
endostatin ranging from 0.01 to 50 μg/ml. All wells received 1 μCi
of [3H]thymidine at the time of treatment.
After 24 h the medium was removed, and the wells were washed with
PBS. Cells were extracted with 1 n NaOH and added to a
scintillation vial containing 4 ml of ScintiVerse II (Fisher
Scientific, Springfield, NJ) solution. Thymidine incorporation was
measured using a scintillation counter. All groups represent triplicate
samples.

Cell Cycle Analysis.

Cell cycle analysis was performed as reported previously
(16)
. Briefly, CPAE cells were grown to confluence in DMEM
containing 10% FBS and growth arrested by contact inhibition for
48 h. A suspension of 500,000 cells was seeded in each well of a
six-well plate in DMEM containing 1% FBS and 5 ng/ml VEGF. Different
doses of arresten were added, and the cells were harvested 18 h
after treatment. Cells were fixed in ice-cold 95% ethanol and
rehydrated 3 h later at room temperature for 30 min in rehydration
buffer (2% FBS and 0.1% Tween 20 in PBS). Next, the cells were
centrifuged at 1,200 rpm for 10 min and resuspended in 0.5 ml of
rehydration buffer. RNase was added at 5 μg/ml and allowed to
incubate for 1 h at 37°C, followed by staining with propidium
iodide at 5 μg/ml. The data were analyzed using a Becton Dickinson
(San Jose, CA) FACStar plus flow cytometer. The percentage of cells in
S phase was calculated using ModFit software.

Endothelial Tube Assay.

Matrigel (Collaborative Biomolecules, Bedford, MA) was added (320 μl)
to each well of a 24-well plate and allowed to polymerize
(17)
. A suspension of 25,000 mouse aortic endothelial
cells in EGM-2 (Clonetics, Inc., Walkersfield, MD) medium
without antibiotic was passed into each well coated with Matrigel. The
cells were treated with arresten, BSA, sterile PBS, or 7S domain in
increasing concentrations. All assays were performed in triplicate.
Cells were incubated for 24–48 h at 37°C and viewed using an Olympus
Optical (Tokyo, Japan) CK2 microscope (3.3 ocular, 10× objective). The
cells were then photographed using 400 DK-coated TMAX film (Eastman
Kodak, Rochester, NY). Cells were stained with Diff-Quik
fixative (Sigma Chemical Co., St. Louis, MO) and photographed again
(17)
. Ten fields were viewed, and tubes were counted and
averaged.

Matrigel Assay.

Matrigel was thawed overnight at 4°C. Before injection into C57BL/6
mice it was mixed with 20 units/ml heparin (Pierce), 150 ng/ml bFGF
(R&D Systems), and either 1 μg/ml arresten or 10 μg/ml endostatin.
Control groups received no angiogenic inhibitor. The Matrigel mixture
was injected s.c. using a 21-gauge needle. After 14 days, mice were
sacrificed, and the Matrigel plugs were removed. Matrigel plugs were
fixed in 4% paraformaldehyde (in PBS) for 4 h at room temperature
and then switched to PBS for 24 h. The plugs were embedded in
paraffin, sectioned, and H&E stained. Sections were examined by light
microscopy, and the number of blood vessels from 10 high-power fields
was counted and averaged.

Inhibition of Tumor Metastases.

C57BL/6 mice were i.v. injected with 1 million MC38/MUC1 cells.
Controls (five mice) received sterile PBS, and the experimental group
(six mice) received 4 mg/kg arresten every other day for 26 days.
Pulmonary tumor nodules were counted for each mouse in both groups and
averaged after 26 days of treatment. Two deaths were recorded in each
group.

In Vivo Tumor Studies.

Human renal cell carcinoma cells (786-0) were maintained in DMEM with
10% FCS until confluent. The cells were harvested, and 2 million were
injected into 7- to 9-week-old athymic nude mice. The tumors were
allowed to grow to ∼700 or 100 mm3. Arresten
was injected i.p. daily at a dosage of 10 or 20 mg/kg. Control groups
received either BSA or the PBS vehicle daily. Human prostate
adenocarcinoma cells (PC-3) were maintained in F12K medium with 10%
FCS until confluent. The cells were harvested, and 5 million were
injected into 7- to 9-week-old male athymic nude mice. The tumors grew
to ∼60 or 200 mm3. The mice were injected daily
with 10 or 4 mg/kg arresten or 20 mg/kg endostatin. Control groups
received daily injections of PBS. In both experiments tumor volume was
measured using the standard formula length × width2 × 0.52
(18)
. Each
group contained five or six mice.

Immunohistochemistry.

Mice were sacrificed after 10–20 days of arresten treatment. Tumors
were excised and fixed in 4% paraformaldehyde. Tissues were paraffin
embedded, and 3-μm sections were cut and mounted on glass slides.
Sections were deparaffinized, rehydrated, and treated with 300 mg/ml
protease XXIV (Sigma) at 37°C for 5 min. Digestion was stopped with
100% ethanol, and sections were air dried and blocked with 10% rabbit
serum. Then, slides were incubated at 4°C overnight with a 1:50
dilution of rat anti-mouse CD-31 monoclonal antibody (PharMingen, San
Diego, CA), followed by two successive 30-min incubations at 37°C of
1:50 dilutions of rabbit anti-rat immunoglobulin and rat alkaline
phosphatase anti-alkaline phosphatase (DAKO, Carpinteria, CA). The
color reaction was performed with new fuchsin, and sections were
counterstained with hematoxylin. Finally, blood vessels in 15 fields
were counted, averaged, divided by the tumor volume, and plotted. For
PCNA staining, tissue sections were incubated for 60 min at room
temperature with a 1:200 dilutions of anti-PCNA antibody (Signet
Laboratories, Inc., Dedham, MA). Detection was carried out according to
the manufacturer’s recommendations using the USA horseradish
peroxidase system (Signet). Finally, the slides were counterstained
with hematoxylin. Staining for fibronectin and type IV collagen
was performed using polyclonal anti-fibronectin (Sigma) at a
dilution of 1:500 and anti-type IV collagen (ICN, Costa Mesa, CA) at a
dilution of 1:100. The Vectastain Elite ABC kit (Vector Laboratories,
Burlingame, CA) was used for detection according to the
manufacturer’s recommendations.

Scatchard Analysis.

Scatchard analysis was performed as described previously
(19)
. Briefly, CPAE cells were plated on a 96-well plate
(10,000 cells per well) in DMEM with 10% FCS and grown to confluence.
The cells were then washed with ice-cold PBS and incubated with 180
pmol of 125I-arresten with and without increasing
concentrations of unlabeled arresten ranging from 150 pmol to 100 nmol
comprising a total of 27 data points. The cells were incubated with
this mixture for 2 h at 4°C. Then, the cells were washed with
ice-cold PBS and extracted with 1 n NaOH, and radioactivity
was measured in a scintillation counter.

ELISA for HSPG.

Direct ELISA was performed as described previously
(9)
.
HSPG (100 ng; Sigma) was coated on a 96-well plate in triplicate in a
2-fold molar excess of binding proteins arresten, bFGF, and BSA.
Binding was established with antibodies to bFGF, arresten, and BSA. The
ELISA was developed with an alkaline phosphatase secondary antibody and
read in a plate reader at absorbance of 405 nm.

Cell Adhesion Assay.

Ninety-six-well plates were coated with human arresten or human type IV
collagen (Collaborative Biomolecules, Bedford, MA) at a
concentration of 10 μg/ml or human vitronectin at 0.5 μg/ml
overnight at 37°C. The remaining protein binding sites were blocked
with 10% BSA (Sigma) in PBS for 2 h at 37°C. HUVECs were grown
to subconfluence (70–80%) in EGM-2 MV medium (Clonetics). The cells
were gently trypsinized and resuspended in serum-free medium (1.5 ×
105 cells/ml). The cells were then mixed with 10μ
g/ml antibody and incubated for 15 min with gentle agitation at room
temperature. Next, 100 μl of the cell suspension were added to each
well, and the plate was incubated for 45 min at 37°C with 5%
CO2. Unattached cells were removed by washing
with serum-free medium, and attached cells were counted. Control mouse
IgG and mouse monoclonal antibody to the humanβ
1 integrin subunit (clone P4C10) were
purchased from Life Technologies. Monoclonal antibodies to theα
1 integrin subunit (clone CD49a), theα
6 subunit, the αV
subunit, andα
vβ3
(LM609) were purchased from Chemicon International (Temecula, CA).

RESULTS

Human arresten was produced in E. coli using a
bacterial expression plasmid, pET-22b (capable of periplasmic
transport, thus resulting in soluble protein) as a fusion protein with
a COOH-terminal 6-histidine tag. The E. coli-expressed
protein was isolated predominantly as a soluble protein, and SDS-PAGE
analysis revealed a monomeric band at 29 kDa. The additional 3 kDa
arise from polylinker and histidine tag sequences and were
immunodetected by both arresten and 6-histidine tag antibodies (Fig. 1, a and b)
⇓
. Human arresten was also produced as a
secreted soluble protein in 293 embryonic kidney cells using the pcDNA
3.1 eukaryotic vector. This recombinant protein (without any
purification or detection tags) was isolated using affinity
chromatography, and a pure monomeric form was detected in the major
peak by SDS-PAGE and immunoblot analyses (Fig. 1, c and d)
⇓
. In addition, human arresten was isolated from human
placenta by gel filtration, HPLC, and affinity chromatography
techniques; a 26-kDa molecule was detected by SDS-PAGE and immunoblot
analyses (Fig. 1, e and f)
⇓
.

In assays of endothelial cell proliferation, a dose-dependent
inhibition of bFGF-stimulated endothelial cells was detected, with an
ED50 value of 0.25 μg/ml (Fig. 2a)
⇓
using E. coli-produced soluble protein. These
results support earlier observations that α1
and α2 type IV collagen isolated from the
Engelbreth-Holm-Swarm mouse sarcoma tumor may be inhibitory to
capillary endothelial cells
(5)
. No significant effect was
observed on the proliferation of renal carcinoma cells (786-0; data not
shown), prostate cancer cells (PC-3) or HPECs, even at arresten doses
of up to 50 μg/ml (Fig. 2, c and d)
⇓
. In
contrast, endostatin inhibited CPAE cell proliferation with an
ED50 value of 0.75 μg/ml, 3-fold higher than
arresten, and did not inhibit A-498 cancer cells (data not shown; Ref.
15
). Cell cycle analysis was also performed using FACScan
technology to assess the antiproliferative properties of arresten in
the presence of VEGF. We observed a decrease in the number of CPAE
cells in S-phase in the presence of arresten. These results correlate
with thymidine incorporation proliferation assays described above
(Fig. 2b)
⇓
.

Inhibition of endothelial cell proliferation. CPAE
(a and e) cells and control
nonendothelial cells, PC-3 cells (c) and HPECs
(d), were treated with concentrations of arresten or
endostatin ranging from 0.01 to 50 μg/ml. All wells received 1 μCi
of [3H]thymidine at the time of treatment. Thymidine
incorporation was measured using a scintillation counter. All groups
represent triplicate samples. b, cell cycle
analysis. Growth-arrested CPAE cells were treated with concentrations
of arresten ranging from 0.1 to 20 μg/ml. The cells were stimulated
with 5 ng/ml VEGF, trypsinized, and harvested after 18 h. The
VEGF (−) value is the percentage of cells in S-phase at
the beginning of the experiment. f–h, endothelial tube
assay with mouse aortic endothelial cells. Ten fields were viewed, and
tubes were counted and averaged (f). Well-formed tubes
can be observed in g treated with 7S domain control
(magnification, ×100). Arresten-treated (0.8 μg/ml) mouse aortic
endothelial cells (magnification, ×100) are shown in
h.

To test the in vivo effect of arresten on the formation of
new capillaries, we performed a Matrigel plug assay in mice
(21)
. Matrigel was placed in the presence of bFGF, with or
without increasing concentrations of arresten. A 50% reduction in the
number of blood vessels was observed at 1 μg/ml arresten and 10μ
g/ml endostatin (Fig. 3a)
⇓
. Collectively, these results suggest that arresten affects
the formation of new blood vessels by inhibiting more than one step in
the angiogenic process.

Matrigel plug assay. Before injection into C57BL/6 mice,
Matrigel (Collaborative Biomolecules) was mixed with 20 units/ml
heparin (Pierce), 150 ng/ml bFGF (R&D Systems), and either 1 μg/ml
arresten or 10 μg/ml endostatin. Control groups received no
angiogenic inhibitor. After 14 days, plugs were removed, sectioned, and
H&E stained. a, sections were examined by light
microscopy and the number of blood vessels from 10 high-power fields
was counted and averaged. b, inhibition of tumor
metastases. C57BL/6 mice were injected i.v. with 1 million MC38/MUC1
cells. Controls (five mice) received sterile PBS, and the experimental
group (six mice) received 4 mg/kg arresten every other day for 26 days.
Pulmonary tumor nodules were counted for each mouse in both groups and
averaged after 26 days of treatment. c–f, in
vivo tumor studies. 786-0 cells (2 million cells) were injected
s.c. into 7- to 9-week-old male athymic nude mice. The tumors were
allowed to grow to ∼700 mm3 (c) or ∼100
mm3 (d; each group contained six mice).
Arresten was injected i.p. daily (10 or 20 mg/kg) for 10 days in
sterile PBS. The control group received either BSA or the PBS vehicle.
e, human prostate adenocarcinoma cells (PC-3) were
harvested and injected s.c. (5 × 106 cells)
into 7- to 9-week-old male athymic nude mice. Experimental groups were
injected i.p. daily with arresten (10 mg/kg) or endostatin (20 mg/kg)
protein. The control group received PBS each day. f,
this experiment was identical to the above PC-3 model, except the
arresten dosage was reduced to only 4 mg/kg/day. The treatment was
stopped after 8 days (arrow); however, significant
inhibition continued for 12 more days with no additional arresten
treatment. At this point, tumors escaped the effect of arresten (data
not shown). g–l, immunohistochemistry. Mice were
sacrificed after 10–20 days of arresten treatment. Tumors were
excised, and 3 μm-sections were mounted on glass slides. CD-31
staining of blood vessels is shown in a control mouse
(g) and an arresten-treated mouse (h).
i, CD-31 blood vessel quantification for
arresten-treated and control-treated tumors. For PCNA staining, tissue
sections were incubated for 60 min at room temperature with a 1:200
dilution of anti-PCNA antibody. Detection was carried out according to
the manufacturer’s recommendations using the USA horseradish
peroxidase system. The PCNA staining is shown in j
(arrows). Staining for fibronectin and type IV collagen
was performed using polyclonal anti-fibronectin at a dilution of 1:500
and anti-type IV collagen at a dilution of 1:100. The Vectastain Elite
ABC kit was used for detection according to the manufacturer’s
recommendations. Fibronectin staining is shown in k
(arrows), and type IV collagen staining is shown in
l (arrows).

To assess the effect of arresten on metastasis, 1 × 106 MC38/MUC1 cancer cells were administered by
tail vein to C57BL/6 mice
(22)
. Treatment with 5 mg/kg
arresten (i.p.) was initiated the following day and continued every
other day for 26 days. The results show a significant reduction of
pulmonary nodules in arresten-treated mice compared with the control
group (Fig. 3b)
⇓
.

Next, we tested the effect of arresten on established primary tumors in
mice. Arresten, E. coli produced, inhibited the growth of
large (Fig. 3c)
⇓
and small (Fig. 3d)
⇓
renal cell
carcinoma tumors. In experiments performed with PC-3 human prostate
tumors in mice, arresten at 10 mg/kg inhibited tumor growth similar to
endostatin at 20 mg/kg (Fig. 3e)
⇓
. A similar degree of
inhibition was observed with arresten administered at 4 mg/kg, and this
inhibition continued for 12 days after arresten treatment was stopped
(Fig. 3f)
⇓
. After 12 days, the tumors escaped the effect of
arresten and began growing at the same rate as the controls (data not
shown). A CD-31 staining pattern of treated (Fig. 3h)
⇓versus control (Fig. 3g)
⇓
mice is shown. Blood
vessels in 15 high-magnification fields were counted and averaged. This
number was divided by the volume of the tumor and averaged
(18.7 ± 6.2 control versus 10.5 ± 7.2 treated; Fig. 3I⇓
). Finally, tumor sections were
stained for PCNA, fibronectin, and type IV collagen. We found no
difference in tumor cell proliferation or in type IV collagen and
fibronectin content surrounding tumor cells in the treated and
untreated mice, again demonstrating the endothelial cell specificity of
arresten (Fig. 3, j–l⇓
, representative arresten-treated
sections).

To gain further insight into the anti-angiogenic mechanism of action of
arresten, we studied its binding to endothelial cells. Iodinated human
placenta arresten was incubated with CPAE cells, and a Scatchard
analysis was performed
(19)
. Our data revealed two
different binding sites (Fig. 4a)
⇓
. The high-affinity, low-capacity binding site has a
Kd1 value of 8.5 × 10−11m and a
maximum number of binding sites of 3 × 106 sites per cell. The other low-affinity,
high-capacity binding site has a Kd2
value of 4.6 × 10−8m and a maximum number of binding sites of
6 × 107 sites per cell. It has
been shown that HSPG binds the α1 NC1 domain of
type IV collagen
(23)
. Also, recent studies have
speculated that α1β1
and α2β1 integrins bind
to type IV collagen isolated from the Engelbreth-Holm-Swarm mouse
sarcoma tumor
(24)
.

Scatchard analysis. a, binding analysis of
arresten to endothelial (CPAE) cells. There are two curves represented
showing high- and low-affinity arresten receptors. b,
HSPG direct ELISA. HSPG was coated on a 96-well plate, and binding to
bFGF, arresten, or BSA was assessed as described in “MATERIALS AND
METHODS.” c–e, cell adhesion assay. HUVECs were
preincubated with an integrin antibody and plated on arresten-coated
(c), collagen type IV-coated (d), or
vitronectin-coated (e) plates. The amount of cell
binding was compared with the control (c), which is
HUVECs incubated with a control mouse IgG. We observed an inhibition of
60% in cell adhesion for the α1 subunit and a 70%
inhibition for the β1 subunit (c and
d).

We assessed the capacity of arresten to mediate endothelial cell
binding via α1β1 andα
2β1 integrins. Our
results show that functionally blocking α1 andβ
1 integrin subunit antibodies significantly
diminish the binding of HUVECs to arresten-coated culture wells (Fig. 4c)
⇓
. We found an inhibition of endothelial cell attachment
to arresten-coated plates of 60% with α1
antibody and 70% with β1 integrin antibody.
The control α6 integrin antibody showed no
binding inhibition to arresten. Theα
Vβ3 antibody did not
inhibit endothelial cell binding to arresten but increased binding
(Fig. 4c)
⇓
. On the other hand, with type IV collagen-coated
plates, we observed an inhibition of 30% withα
1, 40% with β1, and
15% with αvβ3
neutralizing antibodies (Fig. 4d)
⇓
. Again, theα
6 neutralizing antibody had no effect on
binding. We speculate that the difference in cell adhesion between
arresten and type IV collagen-coated plates in the presence ofα
1 and β1 integrin
antibodies is due to additional integrin binding sites on the entire
type IV collagen molecule in comparison with arresten, which may
contain a single integrin binding site (Fig. 4, c and d)
⇓
. To demonstrate the efficiency of theα
Vβ3 neutralizing
antibody, we performed a control adhesion experiment with its ligand,
vitronectin (Fig. 4e)
⇓
. The neutralizingα
Vβ3 andα
V antibodies were able to inhibit endothelial
cell binding to vitronectin by 60 and 90%, respectively.

HSPG binding to arresten was assessed by ELISA. ELISA plates were
coated with HSPG and incubated with arresten, bFGF, or BSA. Our results
show that HSPG binds both arresten and bFGF as reported earlier (Ref.
23
; Fig. 4b⇓
). Taken together in conjunction
with earlier reports
(23)
, these results suggest that
arresten may be binding HSPG on the cell surface (Fig. 4, a and b)
⇓
.

DISCUSSION

We propose that the molecular mechanism associated with the
tumor-suppressing activity of arresten as well as the specific
inhibition of endothelial cell proliferation and migration by arresten
may be mediated by theα
1β1 integrin. These
results suggest that binding of arresten toα
1β1 may down-regulate
VEGF-induced proliferation and migration of endothelial cells, as
suggested previously by VEGF-induced expression ofα
1β1 integrin on
endothelial cells
(25)
.

In support of our findings, it has been shown thatα
1 integrin neutralizing antibodies can
suppress angiogenesis in vivo(24)
. Among the
collagen integrins,α
1β1 activates the
Ras-Shc-mitogen-activated protein kinase pathway, promoting cell
proliferation
(26)
. Our studies suggest that arresten may
be antagonizing this effect in endothelial cells. In addition, Pozzi
et al.(27)
recently described decreased
angiogenesis in tumor-bearing α1
integrin-deficient mice.

Whether arresten functions by suppressing the activity of VEGF and/or
bFGF directly remains to be elucidated. Future comparative studies with
other recently discovered inhibitors such as restin, troponin 1,
kringle 5, pigment epithelium-derived factor, and vasostatin
will also be very insightful in establishing the unique anti-angiogenic
property of arresten
(28,
29,
30)
.

Acknowledgments

Footnotes

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